Onderstepoort ./ownol of TletcrllWI)J Scienre and A111:mal lnd/ustry, Tlolu111e 14, .Y11mbers 1 nnd '2, January and dznil, 1940.
Printed in the Union of South .-\ frica by the Uovei'nm3nt Printer, Pretoria.
Che mical Blood Studies VIII.
A Rapid Spectroscopic Method for (A) The Quantitative Determination of Haemoglobin in Blood and (B) Its Application for the Quantitative Estimation of Hae- moglobin in Milk, Urine, Serum or Plasma and Faeces.
H_,. U. C. S. ROE'l'S, Dcp:utment of Chemical Pathology, Onllerc;tepourt.
T :-; connedion \\·ith :mimal pig·m ent meta holism stwlies the ;mthor :tncl his collaborators nee1led a r~q>i<1 aud eJii1·ient method for cstinta- ting haellloglohin quantitatiH·ly in l>iological JIJ<1t(·riab. ln this communi<:ation ~uch a method is recorded, l1v ,,·hich the blood haemo- globin :mel haemoglobin present in milk, urine, serum o1· plasma and faeces ean be quantitatiYPl)· estimated.
A.- TI-IE QT:"AN'I'Il'ATLVE DETEIUIIINATION OF H.-\E~LOGLOTUN IN HLOOJJ.
Jf etlwds cons,dcred.
Kewcomer, H ::-;_ (J 919) proposed :t haemog-lobinometer, with a
!;olourer1 glass clisl' as a staw1ard for the quantiLativc determination of haemog-lobin as acid haematin. ln 1923 h1· a1hocated the use of a modified Duhoscq colorimeter. 'l'bi,; lll('t hod, lzuown. :1s the :lc\\·l:mner <1isr· Jnclhod, is in cOJllDHlll use. Coheu, B <tllcl. Smith, A. H. (l!Jl !)) used an Autenricth-Hellig·e or Duboscq colorimeter for tnatcltiug- tlte colour against that of a st:Jndan1 a1·id hcl!'matin solution.
AnothN techtt iquc \\·as rlevrlope1l hy \'iT ong, S. Y. (Hl:22) l>y
\\·hi!·lt the iron pl-<'sent- in ihe hloor1 i~ !ld erllline!l <tnrl fro111 this the total amount of haemoglohin is ca lc-ub t-ecl., the assumption being- that iron, npart h !l l i l that contained. in the hnellloglohi11 molecttle is ]Jreseut ouly in tra1·es in bloorl. In Hl'28 he rlPscribe11 ho\\· the imn of the h:1enwg·lohin molecuh• !'<Ill l1e r1etachc!1 hv the action n£
sulphuric acid, the clecompo~ition being fncilit-aterl. with the aid of potassium persulphate. The .intelfering prot0ins are completel:v pre1·i- pitatcrl bY tungstic acid anrl filterecl oFf. Colo1 imetric comparisons
:~remade <lgainst a stanchrd iron solution.. Andes, J. E. and ~orthup,
D. \V. (1938) made use of a p hotoelectrie co lori 111 eter for the com p:tri- sons. Hnnzal, B. F. (1933) determined the inm h~- digesting blood
451
CI-IE~UCAJ, TILOOD STUDIES.
11·ith sulphuric acid and hydrogen peroxide over a mierolnuner, and by comparing the characteristic eolour obtained with thioglycolic
;tcicl and ammonium hydroxide against that of a standard iron solution similarly treated. The laf>t mentioned method was preferred by the author on account of its extreme sensitivity and the elimination of the filtering- process. }Jyers, Y. C. and Eddy, H. :M. (1 939) described slight mo(lific-ations to permit its use ·with 0·05 c.c. of blood.
Denes, A. (1932) esta hlishecl a spectrophotometric method for measuring the 555 · 8 p. p. :mel 527 ·1 p. p. haemochrolllogen absorption bands ratio. From the extinction coefficients of these iwo wave- lengths of the spectrum and the absorption ratio, the haemo).dobin content of the blood eau be calculatetll. The haemin content of liver catalase preparations was determined by Stern, K. G. (1937) by converting the haemin into pyridine haemochromogen and com- paring- the intensity of the absorption bands against that of a standard p1 epared from crystalline blood haemin. 'J'he same principle had beeu used by Z:eile, K. and Hellstri"im, H. (1930) and also by Keilin, D. an(l Hartree, E. F. (1936).
The intensity of the 555 absorption hand of Pyridine haemu- chromogen is made use of in my method ; a Zeiss pocket spectroscope and the prineiple of dilution as desct·ibecl by Riming-ton, C., Roets, G. C. S. and Fourie, P. J . .T. (1938) in the quantitative determina- tion of roproporphyrin being used.
P1"r!Cedure.
Stocl.: Solut·ion.- Di. solve ll.G · G mg. pure recrystallised haemin in 40 c.e. pyridine and dilute up to 100 c.c. with ·lN NaOH solution.
5\tandm-d.- A dilution of 0 · 5 c. c. stock solution up to 100 c.t·.
,,·ith ·1N :XaOH ReTYes a& a standard solution.
Blood.--Tmnsfer 0·4 c.c. oxalated blood into a 200 c.t.:.
measuring flask, add ·1X NaOH up to the mark, shake thoroughly and leaYe at room tempernture for 10 minutes.
Com,zJarison.- Take two similar thin glass test tubes 1· 5 em. in diameter, and measure off 5 c.c. of the standard solution into the one :mel !) r-.c. of the blood solution into the otheT. To each of the tubes add 1 c.c. pyridine and a few mg. sodium hydrosulphite.
Compare the intensity of the 555 absorption bands and dilute the solution showing the more intense band (whether standard or unknown) \Yith a ·1K N aOH solution by means of a calibrated standard pipette until equal intensity is reached in both tubes. After each dilution tlw solution shoulrl he thoroughly mixed by lateral shaking.
Calculations.
)f. wt. of haemog-lobin= GG,800.
'l'he haeni.og·lnbin molecule has four haematin molecules [see Hawk atHl Bergeim (1931)].
Haemin= C31H""01N.1 FeCl.
452
G. C. S. I!OETS.
'L'Itu::; the conc-ent.ratiun haelllogiobin equiY;dellL to the sbnclard
(i6800 ·5
soJutioll= llG·G X 651_
329x+ X
100 mg. per 100 u·.
= J.f·U,J mg. or 0 Ol.fU;) g. per 100 <:.c .
.lu lht· Llll kn(l\l"ll, the amoullt of haemoglobin expressetl as g-rams haemoglobin per 100 c.t.;. blood
,. 200
(i X Q-4. X 0·01-tU0
.r. \"olun1e in e.c. Lo whid1 thr \lllkJJOII"H is tlilntcd
(j 200 -
or = - X 0-
1 X 0 ()1-~D-J
y •.
where JJ= \'oluml' in c.c. to ,,·hich the sLtndanl is tlilutetl.
Pn·coutions.- 'l'he addition of too much sodium hydrosulphite pru(lu(·t•s ;I slig·h! turbitlity, IYhi('h JJW.1· 1·esulL in too high V:llul's.
\\'iUt t•xperience the exa<·t qu;1ntit.1· ncr·ess;tr.\· r;IJJ r•;tsily bt· judged . .l'Jt•sh stot·k solutions ,buultl be made up nwnthl.1· aucll;epL in the tbrk sint·t• tlt•t·ompositioll tnkes place. Stantlanl solutions should be nwtle up daily.
Disc?tssion.
As a clwck on thi;; nwthod simult;tnt•ou;; d<•tenninations ,,·ere nwcle by the .iron method n.~ th'scribed by Hanzal and by the Ne11·comer llisr metlwd. The 1·esrilt~ nhtainetl b.1· the fonlJPr comp;1retl quitf' sutisbd ori h· 11·hilst :hosP of (he last mentioned method did uot. Andes ::tncl :Xort ln;p (lU:JS) hacl tiLt· smnr ex]wriPIICP 11·hen <·omprning the results oht:tiJJecl h,,- \Vong-'s method ;w<l thP ~e\\·t·nmcr diRe method.
'.l'he following· bble sho11·s l'omparatiYf' results obtainerl hy thPse three metlwtls.
~~=-~ --- ---
Animfl.l Spe<·ies.
Sheep normal ........ . Rovine normal.
Bovine normal . ............ . J3o,·in.e normal .. .
Bo,·inc normal ....... . Ho,·inc normal .... .
Hcn·ine normal ........... . :Bovino normal ............ .
Bo,~inc normal ... .. . Bovine <tnn plasmosis ...... . Bodnc ana plasrnosis ......... .
'.l'AHLE [.
Iron :\I cthod. i->pcclroscopic Newcomer Disc :\Jet hod. :\Jet hod.
g. Hb. per g. Hb. per g. Hh. per 100 r·.c. Blood. 100 <·.e . .Blood. 100 c.<·. Blood.
l2. 61 11·47 11·7H
ll·~H 11·~H
1:2· JO l;). 71 J2 · JO 10·!)2
~·I~
6·:2:!
L:Z. ;)/
11.4:2 Jl. 4:2 11 ·8:2 ll·8:2 12·37 J+· 9:3 12 ·4li 10. 5l!
~-08
6· 8:)
1+ .;J.[
12·61 l:l-06 14·04 1:1·78 lii·18 16·87 12·61 10·87 6. ll
!i·08 - - - --- ·- --·- -·--·--- - -- - -
45 3
CHB1HCAL BLOOD STUDIES.
vVith the necessary practice and sufficient equipment 70 to 100 determinations call easily be made during an eight hour working day.
The accuracy and simplicity \vill undoubtedly be increased with a more efficient spectroscopic apparatus. The author attached a Zeiss microspectroscope ocular with a comparison prism to a micro- i'wope and transferred the standard solution to a tube in front of the comp:uison prism which was illuminated ''"ith a mirror in a fixed position. '.L'he stratum of the standard in a little beaker on the microscope stnge ,,·as adjnsted by addition until equal intensity in both spedrn \\":IS n•:~rhecl. The unknown \\·as no"· diluted with known quantities and replnced in the tube in front of the comparative prism until equnl intensity \Yas once more reached. Better results 11·ere not obtained. Similar npparatuses for comparing spectra intensities "·ere designed hy Stern, E.:. G. (1937) and Keilin, D.
and Hartree, K F. (1936) where the strata of liquids could be alteren by movable plung·ers similar to the prin<"iple employed in ordinary colorimeters.
(B) THE AI'PLICATTOJ\" OF TfiJ s :PYJllDINE HAEMOCIIJWMOGEK .YIETHOD TO THE QuAKTITATIVE EsTDIATIOK oF HAE1IOGLOBIN IK MILK, URINE, Smrnr OR :PLAS::IIA .·1::\D FAECES.
;11 etl10ds Considered.
K umemus metholls for the qualitative detection of blood in biological materials can be traced in physiological and biochemical text books like those of Hawk, P. B. and Bergeim, 0. (1931), Plimmer, R. H. A. (1918) and vVhite, D. S., and Fischer, P. (1920).
These methods can be classified into four groups : -
(1) PeTotcidase Reactions.- Haemoglobin reacts upon peroxide giving off oxygen. Oxygen thus liberated gives marked colour reactions with substances of the follo"·ing t~·pe : -
(a) Guaiacum- blue.
( lJ) Aloiu- purple-l'fHl.
(c) Benzicline -Ghte-!l:reen.
(d) :Malachite green- green.
(e) p-Pheny lenediamine hyllrochloride- brown-red.
(f) Phenolphthalein- red.
(g) Ortho-toluidin- bluish.
'l'hese tesi.s, with all their modificatiOns, are based on the same property of haemoglobin. Thus any substance which liberates oxygen in the same \Yay as haemoglobin, will give a positive reaction.
In practire, therefore, pus, milk, faeces, etc., very often give a positive result in the absence of haemoglobin.
(2) The microscozJic 'identification of e1·ythrocytes.-This method is one of the best when non-haemolysed blood is present, for then liquid materials such as urine and milk can be centrifuged and the sediment examined microscopically for erythrocytes.
-:!::)-!
G. C. S. !WETS.
(0) The com.:e1sion of lweuwglouin ·into haemat·in.-(a) In the potassium hydroxide test (Hellet) the reel colour of haematin is made use of to detect the presence uf blood. This test is Yery unsatisfactory since many other ,;ubstances like porphyrins, for example, may con- tribute tr, a red colour of the solution.
(6) 'l'he crystals of hae1uatin can easily be pn~pared by the acetic acid method and microscopically i(lentified. Thi~ is a specific test but the very small quantities to be dealt with in practice makes this method often impracticable.
( 4) SpectToscopic e:t:rl1111 nu tion.- The ;-;pe!'t roscopic examination of tr:-tnslucent mai:erials like lyn1ph, ;-;enuu. pbstua or urine is one of the most sntisfactory '"nys by "hirh hacrnog·lobin cnu be detected in suc:h m:~teri:-tls.
The me(bod !lescrilJed in tlti;-; ,utide lllukes it possible to detect
he~.en10globin in concentration~ as lo11· as -± mg. haemoglobin per 100 c.c. liquicl, on account of the ex(rPJilf' intpnsit:v of the 555 bancl of pyridine haernochromogen.
Procedure.
(1) .lhl/~.-Shake the 1nilJ, sample thorough!~· to obtain an even distribution of erytlHocytes, 11·hich might han graYitatecl to'il·ards the bottom of the container on standing. Trr~nsfer 30 c.c. to a cali- bratecl centrifuge tube, add a fe"'· mg. sapouin, stir well with a glass rocl and leave al. roon. temperature fo1 30 minutes. Aclcl 0·3 c.e. of strong hyclrochlDl'ic, stir well and centrifuge for 15 minutes. Note the volume of 1he liquid. )Ic':lsme oft 10 c.e. of this liquirl into a centrifuge tube, add 2 c.c. of pyridine, mix well and centrifuge for 10 minntes. 'l'o (i e.c. of the ~upernaiant liquid is anded sodium hydrosulphit·e. 'l'he r·omparison is JlO\Y made.
Calculatw11 .
.) 1J
G
X ;_)() X 14 g;J mg. haemoglol1in per l.UO c.c . . ?:=Volume in c.c. to which the unl;no"·n is diluted ..If= Yolumr· in c.•·. of the liqcJicl phfl.'-'r'.
or
6 y
~ X
:30
X 14·l:h mg. haemoglohin per 100 c.c.11·here: i~ thP ·,·olnme in !'.C. to \1·bicl1 tlw mtbwwn is lliluted. 'l'hu,; 30 c.c. of a milk sample containing blood equivalent to 27·41.
mg. haemoglobin per 100 c.c. yielded 25 c.c. of liquid after being centrifuged. 6 c.c. of the final liquid after being treatecl with pyridine and centrifuged had to he cliluterl np to 13 c.c. to match the standard.
13 25
Haemo!!lobin '' found~ J-± · ~);3 . x -6 X 30 - JJJO' , . 1wr l 0(! c.c.
=26 09 mg. per 100 c.c.
-+05
CHE.:Iil< 'AL IlLOOn S'l'1:JJI ES.
The haemoglobin of the erythrocytes in fresh milk can be determined as follows: Centrifnge a Jmo,Yn Yolume of the sample for 30 minutet>, decant off the liquicl carefully, lake the sediment with a 0 · 05 per cent. saponin solutian (1 to 6 c.c. or more) and perform the test as above.
It was observed that the haemoglobin concentrations decreased in the course of time. The following experiment was therefore carried out:-
2 · 5 c.c. of a solution containing 4 · 9G g. haemoglobin per lOU c.c.
was mnde up to 500 c.c. with milk. This mixture 11·as thoroughly shaken. 250 c.c. was exposed in an open flask at room temperature and 250 c.c. was stored in an open flask in the ice chest. Tests were carried out on these hYo samples and it was found that the haemo- globin concentrations decreased equally in both flasks. Table II shows the percentage decrease at different periods.
Time in ho1t1·s. 2 5 24 72 96
'l'AliLE II.
Percentage decrease. 3·5
24·5 62 74·5 80·4
It is therefore necessary to examine milk within two hours after withdrawal from the udder to avoid too large an enor. Furthermore the rate of haemoglobin destruction may vary considerably in different milks, whether from normal or diseased udders, and this point merits further investigation.
(2) Urine and serum (or ZJZasma).-Measure off 10 c.c. into a centrifuge tube, add 2 c.c. of py1idine, mix 'vell and centrifuge for 10 minutes. The test is performed on f: c.c. of the clear supernatant fluid as preYiously described.
Calculations.
14·95 x
i
mg. per 100 c.c.'\Yhere ,?J= Volume in c.c. to 'vhich the unknown IS diluted.
or 14·95 x
t
mg. per 100 c.c.where y =Volume in c.c. to which the standard is diluted.
The haemoalobin concentration in a normal human urine to which blood hall been added, remained ro11stant within the limits of experimental error, for at least 5 hours, but decreased by 34 per cent.
and GG·9 per cent. after 2 and 7 days respectively._ The haemoglobin concentration in urine should therefore be estimated as soon ns possible after collection.
456
G. C. S. JWETS.
(3) Faeces.-Grind iu a mortar 5 grams fresh faeces with 30 c.c.
of a 0 · 05 per cent. saponin solution, leave at room temperature for 15 minutes and centrifuge for 15 minutes. Pipette off 10 c.c. of the liquid, acl<l 2 c.c. of pyridine and a pinch of sodium chloride, stir well Gn<l centrifuge :for 15 minutes. The test is performed on 6 c.e.
of this solution.
Calculat·ions.
X 30
14·95 x
6
X 5 mg. per 100 g. faeces.vVhe1 e .1; =Volume in c. e. to which the unknown 1s clifuted.
] 4 . 6 30 00 f
or . ·!:JG x - X 7 mg. per 1 g. aeces,
y 0
where Y= Volume in c.c. to which the standard is diluted. It should be noted that a possible haemoglobin destruction may also take place in faeces, and to investigate this possibility the following experiment was conducted:-
0 · 3 c.c. quantities of a solution of blood m distilled water (4·4t)5 g. haemoglobin per 100 c.c.) were adcled to 5 g. samples of fresh haemoglobin-free bovine faeces; thus the theoretical amount in eaeh sample should be 269 mg. per 100 g. faeces.
Concentration found=284 mg. per 100 g.
After the satnples had been left at room temperature for 2 hours it \vas 268 rng. per 100 g-. A further deerease of 30 per cent. was found after 24 hours. To obtain the most reliable concentration, estimations shoulfl therefOJe he made as soon as possible after sampling, preferably \Yithin 2 hours after defaecation.
In applying this pyridine haemochromogen method for the quan- titative estimation of haemoglobin in milk, urine, serum or plasma and faeces a slight opalescence or other pigments apart from haemo- globin may interfere with the penetration of light to a certain extent.
The pyridine haemochromogen, however, has such an intense absorp- tion band that this defect is not a serious one. It should also be kept in mind that methaemoglobin, which is frequently encountered is also converted into the pyridine haemochromogen.
SuMMARY.
A spectroscopic method for the quantitative determination of the haemoglobin content o:f blood is described. 'fhe haematin is convertecl into pyridine haemochromogen and a comparison of the intensity of the 555 absorption band made against a known standard. Haemo- globin can be qnantitatiYely estimated in milk, urine, serum, pbsma or faeces.
AcKNOWLEDGMENT.
I wish to thank Dr. H. Graf, Head of this Department, :for his interest ancl encouragement in this work.
457
CHEMICAL llLOOn Sl' l!DIES.
LITEilATURI<:
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t:u. ·
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458
